Methods for culturing organoids

ABSTRACT

The present invention provides a method for culturing organoids, the method comprising: a) disassociating unprocessed organoids to produce a cell suspension; b) sieving the cell suspension through a cell strainer to retain a sieved cell suspension containing cells of about 10 μm to about 1 mm in diameter; and c) seeding cells of the sieved cell suspension into a bioreactor in a cell culture medium comprising an extracellular support matrix.

FIELD OF INVENTION

The present invention relates to a method for culturing organoids.

BACKGROUND TO THE INVENTION

Prior to testing new drug candidates in vivo on animals or humans, invitro testing is usually carried using either primary cell cultures orcell lines. However, results from this testing can be unreliable as thecell cultures do not mimic an in vivo system very well. This can lead tosome good drugs being rejected at the in vitro stages and some poordrugs may be progressed to in vivo trials.

Organoids are three-dimensional structures of heterogeneous tissue thatfunction like an in vivo tissue. In other words, these three-dimensionalstructures of tissue mimic an organ better than a traditional cellculture monolayer. Organoids therefore provide an opportunity to createcellular models of disease, which can be studied to better understandthe causes of disease and identify possible treatments.

Organoids are often generated from stem cells, which can bedifferentiated into cerebral, renal, cardiovascular, and other types oforganoids. Organoid technology has also been used to create a model ofhuman colon cancer progression. These organoids may be created fromnormal intestinal cells mutated to transform into cancer cells, or maybe derived from tumour cells per se. Organoids created from tumours havebeen shown to be a good reflection of the original tumour, providingopportunities for improved in vitro drug testing.

However, to date organoids have only been cultured in laboratorysettings, which rely heavily on the technical skill of the technicianinvolved and produce small numbers of organoids. Organoids are thereforenot readily available in large numbers for drug testing. Furthermore,due to the reliance on technical skill in their culture there is littlestandardisation between cultures, meaning that testing carried out ondifferent batches of organoids may not be directly comparable.

There is therefore a need to produce organoids in larger numbers and toproduce organoids of consistent form and function. This will enablewider use of organoids in drug testing and improve the comparability oftest results.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a method for culturingorganoids, the method comprising a) disassociating unprocessed organoidsto produce a cell suspension; b) sieving the cell suspension through atleast one cell strainer to retain a sieved cell suspension containingcells of about 10 μm to about 1 mm in diameter; and c) seeding cells ofthe sieved cell suspension into a bioreactor in a cell culture mediumcomprising an extracellular support matrix. Sieving the cell suspensionprovides a particular advantage in that the size range of the sievedcells can be controlled. For example, selecting a narrow size range ofcells for seeding in the cell culture medium from actively growing cellsleads to the culture of homogenous organoids having a consistentsize/surface area to volume ratio, resulting in reduced variability andimproved quality. Without being bound by theory, it is believed that thesize of the cells/organoids can be optimised to ensure that allorganoids have good contact with the extracellular support matrix, aswell as good nutrient access and O₂ tension. This ensures that all cellsof the organoids develop appropriately and are of high quality, meaningthat larger numbers of organoids are available for various applications,such as drug screening.

DESCRIPTION

The term organoid simply means resembling an organ. Organoids aretypically defined by three characteristics: self-organization,multicellularity and functionality (Lancaster and Knoblich). Thus, thecells arrange themselves in vitro into the 3-dimensional (3D)organization that is characteristic for the organ in vivo, the resultingstructure consists of multiple cell types found in that particular organand the cells execute at least some of the functions that they normallycarry out in that organ. For example, a prototypical organoid, the mouseintestinal organoid, grows as a single-layered epithelium organized intodomains such that it resembles the in vivo intestinal crypt-villusarchitecture, comprising the different cell types of the intestine(enterocytes, goblet cells, Paneth cells, enteroendocrine cells and stemcells) and surrounding a cystic lumen (Sato et al).

As used herein, unprocessed organoids refer to organoids prepared fromprimary cultures of tissue samples that have not been subjected to anysieving or sizing steps during their culture period. Unprocessedorganoids may be isolated from tissue samples including normal andtumour biopsies of tissues including the alimentary canal, the breast,prostate, lung, liver, ovary, pancreas, skin, kidney, brain and testis.

According to the present invention, unprocessed organoids aredisassociated to break them up into (mainly) single cells. After passingthrough a sieve, larger clumps remain on the filter. The filtrate (i.e.the sieved cell suspension) may contain mostly single cells and/or smallclumps of two or more cells, which may be from about 10 μm to about 1 mmin diameter, preferably about 10 μm to about 200 μm in diameter, morepreferably about 10 μm to about 60 μm in diameter. These are then seededin a cell culture medium comprising an extracellular support matrix.Upon further growth in culture, the viable cells divide and becomemulticellular organoids (referred to herein as stage I organoids). Thesecan be “recovered” whole, from the extracellular support matrix andpassed through sieves of various sizes to extract the organoids of thedesired range of sizes from about 20 μm to about 200 μm (stage IIorganoids) as described below.

Dissociating the unprocessed organoids may refer to the process of celldissociation using trypsin, a proteolytic enzyme which cleaves proteins,to dissociate adherent cells from each other and/or a vessel in whichthey are being cultured. When added to a cell culture, trypsin breaksdown the proteins which enable the cells to adhere to the vessel.Trypsinisation is often used to passage cells to a new vessel. As analternative or complementary dissociation technique, chelation agentsincluding EDTA can be used to break cell-cell junctions.

Cell culture media are well known in the art and will be familiar to theskilled person. Typically, cell culture medium comprises amino acids,salts, glucose, and vitamins and may also comprise iron and phenol red.A culture medium suitable for use in the present invention may begenerated by modification of an existing cell culture medium. Forexample, the cell culture medium may be Dulbecco's modified Eagle medium(DMEM) and may comprise one or more additional components such as anutrient mixture (e.g. Ham's F12), antibiotics/antifungals (e.g.penicillin/streptomycin), buffer (e.g. HEPES), glutamine, and n-Acetylcysteine. The cell culture medium may additionally comprise a serum-freesupplement, such as N2Supplement and/or B27Supplement.

The cell culture medium may comprise about 1% to about 99% v/v of theextracellular support matrix, preferably about 5% to about 85% v/v orabout 10% to about 85% v/v of the extra cellular support matrix. Inpreferred embodiments of the invention the cell culture medium maycomprise about 85% v/v of the extracellular support matrix. Reducing thecontent of extracellular support matrix in the cell culture medium canprovide particular cost advantages over the prior art where it istypical to use 100% extracellular matrix. Surprisingly, the presentinventors have found that the extracellular matrix content can bereduced in the method of the invention without compromising organoidgrowth.

Preferably, the extracellular support matrix is a gel-basedextracellular matrix, which may be synthetic or naturally occurring.Additionally or alternatively, the extracellular support matrix may be asolubilized basement membrane preparation. For example, suitablebasement membrane preparations may be extracted from theEngelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumour rich in suchextracellular matrix proteins as laminin, collagen IV, heparin sulphateproteoglycans, entactin/nidogen, and a number of growth factors.Preferably, the extracellular support matrix comprises at least twodistinct glycoproteins, such as two different types of collagen or acollagen and laminin. In embodiments of the invention the extracellularsupport matrix may be MATRIGEL™ (which comprises laminin, entactin andcollagen IV) or CULTREX™ BME (which comprises laminin, entactin,collagen IV and heparin sulphate proteoglycan). Preferably theextracellular support matrix is MATRIGEL™. Alternatively, theextracellular support matrix may be a synthetic matrix comprisingpeptides based on sequences present in fibronectin, collagen and/orlaminin.

The culture medium may be placed on top of the extracellular supportmatrix and can be removed and replenished as required. In embodiments ofthe invention the bioreactor provides a continuous flow of culturemedium, thereby continuously feeding the organoids. The composition ofthe culture medium may be adjusted over time in order to maximiseuniform growth of the organoids.

The cell suspension obtained following disassociation of the unprocessedorganoids is sieved through a cell strainer to retain a sieved cellsuspension as described above. The cells of the sieved cell suspensionmay be single cells or may be two or more cells joined to form anorganoid. The size of the cells or organoids of the sieved cellsuspension can be controlled by the mesh size of the cell strainer. Forexample, the cell strainer may have a mesh size of about to 10 μm toabout 1 mm or about 10 μm to about 500 μm. Preferably the cell strainerhas a mesh size of about 20 μm to about 100 μm, more preferably about 30μm to about 50 μm. In embodiments of the invention the cell strainer mayhave a mesh size of about 40 μm if mainly single cells are required inthe sieved cell suspension. Suitable cell strainers will be familiar tothe skilled person and include PLURISTRAINERS™.

Following sieving of the unprocessed organoids the sieved cellsuspension is preferably seeded into a bioreactor. Traditional(2-dimensional) cell culture typically involves plating isolated cellson a flat surface (such as a Petri dish or tissue culture treated flask)and supplementing the cells with nutrient media. Cells are typicallystored statically at 37° C. with exposure to 5% CO₂. In contrast,bioreactors allow 3-dimensional cell-cell and cell-matrix interactions,as well as providing spatial and temporal gradients of biochemical andphysical signals, and systemic regulation including cross-talk betweendifferent organ systems. Bioreactors thereby allow cells to bedifferentiated into 3-dimentional tissue structures, such as organoids.A key feature of bioreactors is that they provide a dynamic culturesystem, rather than the static culture systems of traditional cellculture. In static cultures mass transport is based on diffusion, andgenerally limits tissue development to thicknesses less than 0.2 mm dueto drops in oxygen tension and increased concentrations of toxicmetabolites. In contrast, bioreactors provide dynamic mass transport,which allows tissue development on a millimetre to centimetre scale.

In embodiments of the invention the bioreactor may be a fed-batchbioreactor or a perfusion bioreactor, both of which create flow ofculture medium to improve nutrient diffusion. Fed-batch bioreactors aretypically supplied with a discrete amount of culture medium that isusually changed at intervals of days. Fed-batch bioreactors can includestirred flask bioreactors or rotating wall bioreactors, both of whichprovide convective flow of medium to enhance nutrient distribution.Stirred flask bioreactors typically use a magnetic stirrer bar to createa convective flow allowing continuous mixing of the medium. Rotatingwall bioreactors provide a dynamic laminar flow of medium. In preferredembodiments of the invention the bioreactor is a perfusion bioreactor.Perfusion bioreactors use a pump system that can perfuse media thoughcells or tissue in a continuous or non-continuous manner. In perfusionbioreactor systems oxygen and nutrients are supplied to the constructinterior by both diffusion and convection. The flow rate can beoptimized with respect to the limiting nutrient, which is mostly oxygendue to its low solubility in culture medium. Perfusion bioreactors canprovide a continuous flow of nutrients to the organoids, which theinventors have found to lead to improved organoid growth.

Perfusion bioreactors can include fed-plate bioreactors or fluidised bedbioreactors. Fed-plate bioreactors will be familiar to the skilledperson and typically comprise a disposable dish typically formed fromplastic, with a lid that has inlet port(s) on one side and outletport(s) on the opposite side through which media is continuously passed.A gel-based extracellular matrix typically covers the base of the dishand contains the organoids. Fed-plate bioreactors can provide a constantflow of nutrients to the organoids, which the inventors have found tolead to improved organoid growth. Without being bound by theory, theinventors believe that use of a fed-plate bioreactor allows theorganoids to be cultured with less extracellular matrix than isconventionally used in the art. The bioreactors can additionally lead toimproved efficiency of the culture process and also provide costadvantages.

The fed-plate bioreactor is preferably a shallow reactor, such as aflat-bed bioreactor. The bioreactor may be from 1 m×1 m, or smaller,such as a 150 mm or 100 mm flat-bed bioreactor. In embodiments of theinvention smaller vessels such as a 6-well multi-well plates may beused. Alternatively, several bioreactors may be connected in parallel orin series and may be stacked to provide arrays of fed-plate bioreactorsin parallel. For example, the fed-plate bioreactor may comprise an arrayof bioreactors stacked on top of one another such that they are fed inparallel by the same by the same pumps delivering the same media.

In embodiments of the invention the bioreactor can provide continuousflow of nutrients to the organoids. The nutrients are typically providedin the form of a liquid feed, such as a cell culture medium, e.g. asdescribed above, and the concentration of components of the medium canbe increased or decreased over time to maximise uniform growth of theorganoids. For example, it is possible to continuously feed at variousdilution rates from a constant concentration liquid or constantly feedfrom a variable concentration feed. In alternative embodiments of theinvention, nutrients may be pulse fed meaning that doses of liquid feedor other components can be added to the bioreactor in discrete amounts,but at a regular frequency. For example, pulse feeding may compriseadministering a discrete amount of liquid feed or culture medium atintervals of 1 minute or 1 hour or 1 day.

Cells of the sieved cell suspension may be seeded into the bioreactor ata concentration of about 20,000 cells/ml to about 10,000,000 cells/ml,preferably about 200,000 cells/ml to about 800,000 cells/ml, morepreferably about 400,000 cells/ml to about 600,000 cells/ml. The cellsmay be seeded into cell culture medium comprising extracellular supportmatrix as described herein. The cell culture medium may additionallycomprise one or more kinase inhibitors, such as a Rho-associated proteinkinase (ROCK) inhibitor. Such inhibitors may enhance the recovery andgrowth of the seeded cells. Kinase inhibitors may be present atconcentrations of from about 0.1 μM to about 100 μM, preferably about 1μM to about 20 μM. In embodiments of the invention the cell culturemedium may comprise a kinase inhibitor at a concentration of about 10μM.

Following seeding of the sieved cell suspension the cells are preferablycultured in the bioreactor to form stage I organoids. Preferably atleast 100,000 or at least 250,000 stage I organoids are generated. Inembodiments of the invention around 500,000 stage I organoids may begenerated. However, the number of stage I organoids generated could bearound 1,000,000 or around 5,000,000 or around 10,000,000 or more. Theorganoids may be cultured for periods of not less than 24 hours or notless than 36 hours. In embodiments of the invention the organoids may becultured for periods of from about 24 to about 96 hours, preferably fromabout 36 to about 72 hours. In preferred embodiments of the inventionthe organoids are cultured for about 48 hours.

Stage I organoids may be recovered from the bioreactor by transferringthe cell culture medium/extracellular matrix mixture to a centrifugetube and centrifuging the tube to retain a gel layer formed by theextracellular matrix. The gel layer may then be incubated with cellrecovery solution and centrifuged to form an organoid pellet.

Cell recovery solutions are well known in the art and will be familiarto the skilled person. The cell recovery solution acts to breakdown theextracellular support matrix to release the organoids without damage.Suitable cell recovery solutions include CORNING® Cell RecoverySolution.

Following removal of the stage I organoids from the bioreactor theorganoids (which may be in the form of an organoid pellet) arepreferably suspended in cell culture medium, which may comprise anutrient mixture such as Ham's F12. The organoids may then be sievedthrough at least two cell strainers having different mesh sizes toobtain a suspension of stage II organoids having a diameter of about 20μm to about 200 μm.

As mentioned above, the size of the organoids can be controlled by themesh sizes of the cell strainers. For example, the cell strainers mayhave mesh sizes of about 20 μm and about 200 μm, preferably about 30 μmand about 100 μm, more preferably about 40 μm and about 85 μm. In moredetail, the suspension comprising the recovered stage I organoids may bepassed through a first cell strainer have a large mesh size (e.g. 85 μm)and all organoids larger than that mesh size may be discarded. Thefiltered contents may then be passed through a second cell strainerhaving a smaller mesh size than the first cell strainer (e.g. 40 μm) andall debris smaller than that mesh size may be discarded. The size of theretained stage II organoids will therefore be determined by the meshsizes of the first and second cell strainers. In the example above, thestage II organoids will be between about 40 μm and about 85 μm indiameter.

The stage II organoids may be frozen for storage or shipping. In moredetail, the stage II organoids may be resuspended in a freezing mediumat a concentration of about 10,000 to about 500,000 organoids per 200 μlof freezing medium, preferably about 20,000 to about 300,000 organoidsper 200 μl of freezing medium, more preferably about 50,000 to about100,000 organoids per 200 μl of freezing medium. The suspended organoidsmay then be frozen at −80° C.

Freezing media will be familiar to the person skilled in the art andtypically comprise a mixture of cell culture medium and dimethylsulfoxide (DMSO), optionally also including foetal calf serum (FCS).Suitable freezing media are commercially available and include GIBCO®Recovery Cell Culture Freezing Medium.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to one or more specificembodiments in which:

FIG. 1 shows a comparison of standard bench-scale culture of organoidsand a fed-plate bioreactor of the present invention. Part A shows the24-plate containing a total of 12 ml of cell culture medium, 1.2 ml ofMATRIGEL™ and 4.8 million cells. Part B shows a 100 mm fed platebioreactor containing 15 ml of cell culture medium, 6 ml of MATRIGEL™ ora MATRIGEL™ media mix and 2.4 million cells.

FIG. 2 shows a schematic illustration of the fractionation process inwhich stage I organoids are sieved through two cell strainers ofdifferent mesh sizes to obtain a suspension of stage II organoids. Thelower right-hand panel illustrates the size distribution of the stage IIorganoids obtained using cell strainers with mesh sizes of 85 μm and 40μm.

FIG. 3A and FIG. 3B show the effect of WEE1 inhibitor on organoidscultured using a fed-plate bioreactor according to the presentinvention. Cultured organoids were fed with 25 μl of growth mediumcontaining 1 to 2.5 μM MK1775, a WEE1inhibitor (8 replicates). FIG. 3A:after 5 days organoids were fixed and stained with Hoeschst (specificfor DNA, i.e. nuclei of eukaryotic cells) and phalloidin (specific forF-actin). The overall shape of organoids in untreated cultures or at lowconcentrations of inhibitor varied from round and cyst-like toconvoluted and branched. As the concentration of inhibitor increases,cells are shed from the outer layer of the organoid, leading to a lossin complexity and a decrease in the overall size. FIG. 3B: morphometricanalysis of organoid parameters confirmed the observations of toxicity.Organoid size decreases with increasing concentrations of inhibitor,while apoptotic nuclei and nuclei roundness (due to swelling beforeapoptosis) both increase with drug concentration.

EXAMPLE 1—COLORECTAL ORGANOID CULTURE PROTOCOL

1. Maintenance of Unprocessed Organoids

All Maintenance Protocols are performed within a Class II laminar flowcabinet to maintain sterility.

3+ Medium contains Advanced DMEM/F12 (with high glucose and pyruvate)supplemented with HEPES, 1×GLUTAMAX™ & penicillin/streptomycin (100U/mL).

6+ Medium contains Advanced DMEM/F12 (with high glucose and pyruvate)supplemented with HEPES, 1×GLUTAMAX™, penicillin/streptomycin (100U/mL), 1×B27, 1×N2 and 1.25 mM n-Acetyl cysteine.

1.1 Manual Trituration Protocol (ISO50, ISO78)

Trituration is carried out using un-supplemented DMEM/F12 media,pre-equilibrated to 4° C. Fresh MATRIGEL™, stored in frozen aliquots, isthawed and maintained in liquefied form on ice.

The culture medium on the organoids in polymerised MATRIGEL™ in a24-well plate is replaced with chilled media. The MATRIGEL™ domes aredisrupted with the end of a 1000 μL pipette tip. The contents of no morethan 3 wells are combined in 15 ml tubes, on ice. Chilled media isadded, to dilute the used MATRIGEL™. Organoids are then pelleted bycentrifugation at 1000 rpm for 3 minutes and the old media and MATRIGEL™is removed by aspiration. The pelleted organoids from several tubes arecombined in a volume of about 400 μl media and disaggregation is carriedout by passing them up and down at least 100 times, through a 1000 μLpipette tip. Chilled medium is added, the organoids are pelleted bycentrifugation and the media is removed by aspiration, leaving a drypellet. The required volume of fresh 100% MATRIGEL™ is added, and themix is plated out at 50 μL per well of a 24-well plate (or as required).After polymerisation of the MATRIGEL™ at room temperature for at least15 minutes, 500 μL of “6+” crypt culture medium is added to each welland the plate is cultured in a humidified incubator at 37° C. and 5%CO₂. The media is changed every 2-3 days until the organoids grow toolarge or dense for the MATRIGEL™ and require repeat disaggregation bytrypsinisation or trituration.

1.2 Trypsinisation (ISO72)

Trypsinisation procedures are carried out using un-supplemented DMEM/F12media and TRYPLET™ pre-equilibrated to room temperature. FreshMATRIGEL™, kept in frozen aliquots, is thawed and maintained inliquefied form on ice.

Organoids in MATRIGEL™ are washed in PBS and then incubated for 3minutes in TRYPLE™ (250 μL per 50 μL MATRIGEL™ dome) at 37° C. Thereaction is stopped by inhibiting the enzyme with an equal volume ofDMEM/F12+10% FBS or Defined Trypsin Inhibitor (INVITROGEN®). TheMATRIGELT™ and media mixture is pipetted up and down 10-20 times througha 1000 μL pipette tip to assist disaggregation. The contents of up to 3wells are combined in 15 ml conical bottomed tubes. DMEM/F12 media isadded, to dilute the used MATRIGEL™, which is aspirated followingcentrifugation at 1000 rpm for 3 minutes. The required volume of 100%MATRIGEL™ is added to the dry organoid pellet and the mix is plated outat 50 μL per well of a 24-well plate (or as required). Afterpolymerisation of the MATRIGEL™ at room temperature for at least 15minutes, 500 μL of “6+” crypt culture medium is added to each well andthe plate is cultured in a humidified incubator at 37° C. and 5% CO₂.The media is changed every 2-3 days until the organoids grow too largeor dense for the MATRIGEL™ and require repeat disaggregation bytrypsinisation or trituration.

2. Bioprocessor Protocols

FIG. 1B shows a 100 mm flatbed bioreactor and a comparison of the samewith a 24 well plate (FIG. 1A).

2.1 The Bioprocessor

Bioprocessing is the process by which organoids are cultured in a 100 mmdish, in a “flat-bed” bioreactor and then separated into different sizesby fractionation (see section 4 ) using 85 μm and 40 μm PLURISTRAINERS™to obtain organoids of the desired dimensions.

The 100 mm dish flat-bed bioreactor, has a lid, specially adapted withinlet and outlet valves to facilitate addition to and aspiration ofmedia from the surface of the organoid/MATRIGEL™ mix contained withinthe dish. Fresh growth media is contained within a “feed reservoir”bottle with an attached HEPA filter and dip tube. An identical bottle isthe waste reservoir. Tubing to these bottles is adjoined to pumpmanifold tubes and attached to a peristaltic pump by tube clips, asappropriate to allow fresh media to be pumped from the media bottle ontothe MATRIGEL™ surface, or waste media to be removed to the wastereservoir. Hence, the system is termed a “Fed-plate” bioreactor as themedia exchange does not need manual intervention.

2.2 Seeding a “Fed-Plate” Bioreactor (ISO50)

All parts of the bioreactor are sterile. Parts are autoclaved orotherwise sterilised by soaking in 70% alcohol, as necessary. 6+ growthmedia, pre-warmed to 37° C. is placed in the “feed” reservoir.

Organoids are trypsinised by incubation with TRYPLE™ according to theprotocol above (1.2 Trypsinisation). Following aspiration of the oldMATRIGEL™ from the combined, trypsinised organoids, the pellet isre-suspended in 10 mL chilled DMEM/F12 and passed through a 40 μm cellstrainer. The resulting filtrate consists mainly of single cells. (Thelarger aggregates caught in the strainer can be harvested and used ifrequired.) A 100 mm culture dish is seeded with 400,000-600,000 cells/mLin a total volume of 6 mL of 100% MATRIGEL™ or a MATRIGEL™:6+ media mix(from 2% to 99.9% MATRIGEL™). Following polymerisation of the MATRIGEL™at room temperature or 37° C., 15 mL 6+ growth medium containing ROCKinhibitor is added. The plate is incubated for 24 hours under staticconditions.

2.3 Using the Bioprocessor

The lid of the 100 mm dish containing the cells seeded in MATRIGEL™ (see2.1 Seeding a “fed-plate” bioreactor) is replaced with the sterile,“fed-plate” bioreactor lid. The bioreactor, media bottles and associatedtubing are maintained in a humidified incubator at 37° C. and 5% CO₂with the pump at 0.9 rpm maintaining a flow rate of 0.59 ml/hr.Organoids are normally cultured for 48 hours prior to recovery,fractionation and freezing (see protocols below).

3.Recovery of Whole Organoids From MATRIGEL™ (or MATRIGEL™/Media Mix)

Sterility should be maintained throughout this process.

The MATRIGEL™/Organoid layer is washed with PBS. 10 ml chilled “CellRecovery” solution (Invitrogen) is added and the MATRIGEL™ layer isdisrupted with the end of a 1000 mL tip. The plate is incubated on icefor 25 min with gentle agitation. The contents of the dish are thenplaced in a 50 mL tube and made up to 50 mL with DMEM/F12. The organoidsare pelleted by centrifugation at 1000 rpm for 3 min. or until anorganoid pellet is visible. The used MATRIGEL™/media mix is aspiratedand the organoids are re-suspended in 5-10 mL of DMEM/F12 prior tofractionation.

4.Fractionation (Sizing Protocol)

The “recovered” organoid suspension (see previous section) is passedsequentially through cell strainers, to retrieve organoids of therequired dimensions. The schematic (FIG. 2) shows the process with 85 μmand 40 μm strainers. An estimate of the numbers and size of theorganoids is obtained in each fraction using the BECKMAN COULTER® MS3,using ISOTON® II buffer with 40% glycerol and a 400 μm aperture. A smallfraction of the filtrates is trypsinised to single-cell using TRYPLE™and counted to give an estimate of the total number of cells within theorganoids and thus an average cell count per organoid.

5. Freezing Protocol for Seeding Organoids into a 384-Well Plate

The organoids pelleted by centrifugation and re-suspended in commercialfreezing mixture such that there are 500,000 organoids per mL, 200 μLper cryovial (100,000 organoids). The cryovials are transferred to a “MrFrosty” container and placed at −80° C. freezer for at least 24 hours.The vials can then be transferred to other containers and storedlong-term at −80° C.

EXAMPLE 2—COLORECTAL ORGANOID VALIDATION

Drug Titration Assay Results

ISO50 (Isolation number 50 ) colorectal cancer organoids were culturedin a fed-plate bioreactor for 3 days, recovered from the MATRIGEL™ andstored frozen at −80° C. They were subsequently revived and used to seeda 384-well plate at a density of 350 organoids per well in 12 μlMATRIGEL™. The organoids were fed with 25 μl growth medium containing0-2.5 μM MK1775 , a WEE1 inhibitor (8 replicates). After 5 days, theorganoids were fixed and stained with Hoechst (blue fluorescent stainspecific for DNA i.e. nuclei of eukaryotic cells) and phalloidin (pinkstain for F-actin).

Confocal imaging shows the blue nuclei of cells with pink-stained actinfilaments (see FIG. 3A). The actin filaments are spread throughout thestructures, but are more concentrated in the lumen, in the centre of theorganoid. Representative organoids are pictured from selected wells ateach concentration. The overall shape of organoids in an untreatedculture, or at low concentrations of inhibitor, can vary from round andcyst-like to convoluted and branched. As the concentration of inhibitorincreases, cells are shed from the outer layer of the organoid, leadingto a loss of complexity and a decrease in the overall size.

Morphometric analysis of over 1000 parameters of the organoids confirmedthe observations of toxicity. Example graphs are shown in FIG. 3B.Organoid size decreases with increasing concentrations of inhibitor.Apoptotic nuclei and nuclei roundness (due to swelling beforeapoptosis), both increase with drug concentration.

Each of the following references is incorporated by reference in itsentirety for all purposes.

Lancaster, M. A.; Knoblich, J. A. (2014) “Organogenesis in a dish:modeling development and disease using organoid technologies.” Science345:1247125-1247125.

Sato, T.; Vries, R. G.; Snippert, H. J.; van de Wetering, M.; Barker,N.; Stange, D. E.; van Es, J. H.; Abo, A.; Kujala, P.; Peters, P. J.;Clevers, H. (2009) “Single Lgr5 stem cells build crypt villus structuresin vitro without a mesenchymal niche.” Nature 459:262-265.

1. A method for culturing organoids, the method comprising: a)disassociating unprocessed organoids to produce a cell suspension; b)sieving the cell suspension through a cell strainer to retain a sievedcell suspension containing cells of about 10 μm to about 1 mm indiameter; and c) seeding cells of the sieved cell suspension into abioreactor in a cell culture medium comprising an extracellular supportmatrix; wherein the bioreactor is a fed-plate bioreactor.
 2. The methodof claim 1, wherein the fed plate bioreactor is a flat-bed bioreactor.3. The method of claim 1, wherein the bioreactor is a perfusionbioreactor.
 4. The method of claim 1 wherein the fed-plate bioreactorcomprises an arrangement of bioreactors that are fed in parallel or inseries.
 5. The method of claim 1, wherein the cell culture mediumcomprises about 1% to about 99% v/v of the extracellular support matrix.6. The method of claim 5, wherein the cell culture medium comprisesabout 5% to about 85% v/v of the extracellular support matrix.
 7. Themethod of claim 1, wherein the extracellular support matrix is asolubilized basement membrane preparation.
 8. The method of claim 1,wherein the extracellular support matrix comprises laminin, entactin andcollagen IV, or comprises laminin, entactin, collagen IV and heparinsulphate proteoglycan.
 9. The method of claim 1, wherein the cellstrainer of step (b) has a mesh size of about 30 μm to about 50 μm. 10.The method of claim 1, further comprising culturing the cells in thebioreactor to form stage I organoids.
 11. An organoid produced by themethod of claim
 1. 12. An organoid produced by the method of claim 8.13. An organoid produced by the method of claim
 9. 14. An organoidproduced by the method of claim 10.